Mint plant &#39;Kushal&#39; for late transplanting

ABSTRACT

The present invention was related to the development of a novel, distinct high yielding plant with rapid regeneration ability obtained through screening of the somaclones in a methodical way for fast regeneration in the tissue culture stage itself which was achieved by inventing the plant ‘Kushal’. The plant yield higher herbage with corresponding high essential oil when evaluated with available superior varieties of mint in late planting condition during April when the fields are vacated after the harvest of Rabi crop like wheat, chickpea, coriander etc. Further the suckers required for commercial vegetative planting can be produced even in low land condition as the plant is reasonably tolerant to water logging compared to the best check ‘Kosi’.

FIELD OF THE PRESENT INVENTION

[0001] The present invention is related to the development of a novelhigh yielding plant obtained through a unique method of screening of thesomaclones in a methodical way for better regeneration. The selectedplant establishes quickly in the field when shoot cuttings are plantedmuch later than the normal planting time/season. The overall essentialoil and menthol yield from the said plant is high compared to otherexisting varieties when planted late through shoot cuttings astransplanted mint. This plant is unique and clearly distinct from allother existing varieties of Mentha arvensis L. The new variety has beennamed as ‘Kushal’ which can be propagated vegetatively through suckersfor commercial cultivation. All the experiment related to thedevelopment of the invented plant ‘Kushal’ were conducted at the farm ofCIMAP, Lucknow, UP, India.

BACKGROUND AND PRIOR ART REFERENCES OF THE PRESENT INVENTION

[0002]Mentha arvensis Linn. var piperescens. Holmes (menthol or Japanesemint) is a highly valued industrial crop due to menthol, which ispurified by crystallization through freezing from its essential oil. AtCIMAP continuous improvement of the genotypes leading to betterment ofdifferent commercially important characters are underway. One of thecritical steps in menthol mint cultivation is the date of planting whichdetermines the optimum yield of menthol after harvesting. In India theplanting duration extends from mid of January to first quarter ofFebruary. Prior to planting the crop during this period, a small portionof land is used for sucker production during the month of September toJanuary. During this period the underground suckers multiply vigorouslywhich are used for planting in the main field.

[0003] But the sucker planting of mint during January and February (thenormal planting time) interferes into the Rabi season crops like wheat,chickpea, coriander etc having a full grown crop stand at that time tobe harvested at the mid to end of April. The Rabi cropping season startsat September-October to April-May. Planting in the month ofJanuary-February thus leads to the compromise for some of the Rabiharvest. The existing varieties of mints if planted late, i.e. inMarch-April show significant reduction in the biomass yield. In thevarietal improvement programmes, the genetic alternations leading toenhancement in the menthol content in the essential oil and improvingother adaptive characters determining the yield and quality of essentialoil are most desirable. So we planned experiments for developing agenotype which can be transplanted through stem cuttings instead ofsucker planting after the Rabi harvest in mid April and also throughsucker mode during normal planting in January-February withoutcompromising on yield. The most desirable trait for such a genotypewould be high and rapid regenerability of cuttings and that is what wasachieved in the plant ‘Kushal’ of Mentha arvensis.

SUMMARY OF THE PRESENT INVENTION

[0004] The object of the present invention was to develop a novel highmenthol producing plant through screening of the somaclones in amethodical way for better regeneration in the tissue culture stageitself which was achieved by inventing the plant ‘Kushal’. The selectedplant adapts quickly to the field condition when shoot cuttings areplanted late than the normal planting duration. The essential oil yieldfrom the said plant is high coupled with the property of being rich inmenthol compared to other existing varieties when planted late from theshoot cuttings. This plant is unique and clearly distinct from all otherexisting varieties of Mentha arvensis L. The new variety has been namedas ‘Kushal’ which can be propagated vegetatively through suckers forcommercial cultivation. The suckers required for commercial vegetativeplanting can be produced even in water logging condition as the plant istolerant to water logging compared to the best check ‘Kosi’.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

[0005] We used the plant ‘Himalaya’ (U.S. Plant Pat. No. 10935), anelite mint genotype for large scale screening of in vitro raised clones(Khanuja S P S, Shasany A K, Dhawan S, Sushil Kumar, 1998, Rapidprocedure for isolating somaclones of altered genotypes in Menthaarvensis. J Medicinal and Aromatic Plant Sciences 20:359-361) to selectclones with high regeneration capacity. Experiments were conducted usingthese clones for in vitro regenerability of shoot explants on MS basedmedium (Murashige T and Skoog F, 1962, A revised medium for rapid growthand bioassay with tobacco tissue cultures; Physiol. Planta. 15473-497.). Passage transfers were repeatedly done at 15 days intervalsfor which every time the appearing shoots (after one month) were cut andinoculated afresh on the medium.

[0006] Out of about 1645 shoots screened 25 shoots showed the initiationof regeneration much earlier (fast growth as observed visually). After20 passage transfers only one shoot (Clone M12), repeatedly demonstratedextra early initiation of regeneration followed by rapid growth comparedto the other screened shoots. The internodal explants from these clonecould show sign of shoot initiation from multiple sites within 7 days ofinoculation compared to other clones showing regeneration much later.During passage transfers of the regenerated shoots the growth was rapidcompared to the other clones as observed visually. The selected shootwas then multiplied in the medium described earlier (A. K. Shasany, S.P. S. Khanuja, S. Dhawan, U. Yadav, S. Sharma, S. Kumar, Highregenerative nature of Mentha arvensis internodes. Journal ofBiosciences 23 (1998) 641-646.).

[0007] The plantlets were hardened, transferred to the glasshouse andsubsequently grown in the field for sucker production. This clone (M12)at this stage was designated as CIMAP/GRB 12. The suckers produced wereplanted in the field for comparative field evaluation in initial fieldtrials with other varieties in the last week of January as normalplanting date for 2 consecutive years in RBD fashion and differentgrowth and yield characteristics were recorded (Table 1). For fieldtrials 10 m×10 m plots were prepared by adding only FYM 1.5 ton per haand the crop was harvested 110 day after planting and the secondharvesting was 70 after the first harvest. TABLE 1 Comparative growthand yield characteristics of plant of invention ‘CIMAP/GRB 12’ inrelation to the existing Japanese mint varieties (Average of fieldtrials in the years 1999 and 2000) when planted during last week ofJanuary (normal planting time). Property Himalaya Kalka Kosi CIMAP/GRB12Plant height 60.9 ± 6.2 51.6 ± 2.7 66.2 ± 3.1  70.9 ± 0.7 (cm) Canopy(cm) 62-70 40-60 65-72 83-85 Leaf number 40.2 ± 4.2 38.0 ± 2.0 50.4 ±3.8 41.0 ± 1.0 Branch length 40.4 ± 6.8   45 ± 9.7 52.0 ± 5.7 46.7 ± 0.9Branch number 29.2 ± 4.0 27.2 ± 1.1 30.4 ± 1.7 32.0 ± 1.2 Leaf length 7.1 ± 0.5  7.4 ± 0.8 7.40 ± 0.7 7.75 ± 0.3 Leaf breadth  4.5 ± 0.2  4.0± 0.3 4.60 ± 0.5  4.8 ± 0.4 Petiole length 1.75 ± 0.3  1.5 ± 0.5  1.4 ±0.3  1.5 ± 0.1 Oil % 0.7 0.8 0.8 0.8 Menthol % 80 82 76 78 Herbage yield1.25 0.80 1.70 1.72 (Q per 100 m²)

[0008] A separate nursery was planted in February for Apriltransplanting and subsequent evaluation. The plants produced wereallowed to grow in the nursery till April and the stem cuttings of 20 to40 cm were transplanted after tilling, pulverizing the land. The overallobjective was to develop a genotype amenable to planting late, with highyield of oil and menthol content through higher herbage production. Theplant was tested in field trial for oil yield, menthol content andherbage production against the checks (varieties developed by CIMAP).

[0009] Replicated Initial field trials were conducted following normalagronomic practices by planting shoot cuttings raised in the nursery inthe month of April, 1999; bench scale field trial during 2000; and pilotscale field trials during 2001, 2002 in RBD fashion and different growthand yield characteristics were recorded. For initial field trials 5 m×5m plots, for bench scale and pilot scale field trials 10 m×10 m plotswere prepared by adding only FYM 1.5 ton per ha. Astonishingly the plantCIMAP/GRB 12 (named as ‘Kushal’ at this stage) was able to out-competeall existing varieties in late planting. TABLE 2 Comparative growth andyield characteristics of plant of invention ‘Kushal’ in relation to theexisting Japanese mint varieties (Average of initial field trials in theyears 1999 for late transplanting) when planted during April. Hima-CIMAP/ Property laya Kalka Kosi GRB12 LSD at 5% Plant 109.7 ± 55.6 ± 2.986.7 ± 4.1 108.0 ± 1.3 19.6 height 5.8 (cm) Oil % 0.61 0.70 0.78 0.780.23 Menthol 80 82 76 78 0.21 % Herbage 3.25 1.42 2.50 3.30 0.60 yield(Q per 100 m²) Oil yield 1.78 1.01 1.75 2.33 0.54 (Kg per 100 m²)

[0010] TABLE 3 Comparative growth and yield characteristics of plant ofinvention ‘Kushal’ in relation to the existing Japanese mint varieties(Average of Bench scale field trials in the years 2000 for latetransplanting) when planted during April. Plant Leaf- Oil Herb yield Oilyield height stem content Q/ha Kg/ha Genotypes cm ratio (%) (estimated)(estimated) CIMAP/GRB12 108.0 0.88 0.78 333 235.0 Shivangi 79.7 1.001.11 143 137.9 Teesta 87.3 0.86 0.80 174 126.9 Saksham 88.0 0.71 0.82249 186.0 Himalaya 109.7 0.80 0.61 322 177.7 Kosi 86.7 0.92 0.78 249174.4 C.D. 5% 19.6 0.37 0.23 62 54.0 1% 26.0 0.50 0.31 83 71.9

[0011] TABLE 4 Comparative growth and yield characteristics of plant ofinvention ‘Kushal’ in relation to the existing Japanese mint varieties(Average of pilot scale field trials in the years 2001 for latetransplanting) when planted during April. Menthol yield Oil yield(kg/ha) (kg/ha) Clones Herb yield (q/ha) (estimated) (estimated)CIMAP/GRB12 328 221 177 Shivalik 236 128 98 Himalya 294 177 140 Kosi 318185 147 CD 5% 62 50 40 1% 92 67 53

[0012] TABLE 5 Comparative growth and yield characteristics of plant ofinvention ‘Kushal’ in relation to the existing Japanese mint varieties(Average of pilot scale field trials in the years 2002 for latetransplanting) when planted during April. Herb Yield Oil Yield (q/ha)(Kg/ha) (estimated) (estimated) CIMAP/GRB12 305 194 Shivalik 238 136Himalaya 270 163 Kosi 299 180

[0013] In all the field trials conducted for transplanted mint,‘CIMAP/GRB 12’, now onwards referred as ‘Kushal’ performed better thanall other mint varieties in terms of menthol and biomass (herbage)yield. The biomass yield of the genotype varied between 305 quintal perhectare (Q/ha) to 333 Q/ha. The menthol yield was in the range of 177kilogram per hectare (kg/ha) to 194 kg/ha. So the plant ‘Kushal’performed better than the best check varieties ‘Saksham’ (U.S. PlantPat. No. 13279) and ‘Kosi’ (U.S. Plant Pat. No. 12426) in all the fieldtrial consistently when transplanted during the month of April (Table 2to 5). This plant ‘Kushal’ also performed at par in total biomass(herbage) yield compared to the best control variety ‘Kosi’ when plantedin the month of February, which is the normal month of planting ofmints.

[0014] Interestingly, another most important character was encounteredby us in the year 2001 when the sucker producing plots fields at CIMAPfarm were submerged due to untimely rains. At the initial stage ofplantation when the fields were submerged with water for a longerduration (more than 7 days) then all other varieties did not regenerateas maximum plants and suckers in the soil were damaged due to lack ofaeration. The sucker fields for other varieties became patchy with poorgrowth where as the plant ‘Kushal’ was not affected in terms of growthparameters. So, experiment was conducted in the next year by logging thefields with water for sucker production and observations (Table 6) wererecorded. As observed from the experiment, the plant ‘Kushal’ canwithstand waterlogging, whereas the survival, growth and proliferationof other varieties were severely hampered leading to less and weeksucker yield. TABLE 6 Observations recorded in the 1 m² area during themonth of September 2002 in the mint variety Kushal (Date 15-9-2002)Primary Survival % Plant height (cm) branches/Plant S. Kus- Saks- Kus-Saks- Kus- Saks- Kosi No. hal ham Kosi hal ham Kosi hal ham 1. 100 50 330 15 10 7 5 3 2. 100 40 20 25 26 15 8 8 4 3. 90 50 28 30 20 10 7 5 4 4.100 50 32 35 15 16 9 6 5 5. 100 30 20 30 17 15 12 7 6 6. 100 40 30 28 2018 10 8 5 7. 90 52 34 30 16 15 6 5 6 8. 92 50 30 35 18 16 7 4 5 9. 96 5236 30 22 12 10 5 5 10. 100 56 40 36 20 15 12 9 4 11. 100 60 30 32 18 178 5 8 12. 94 40 28 26 24 20 12 10 5 S.E.± 1.166 2.311 1.589 0.905 0.9580.829 0.601 0.533 0.354

[0015] Taxonomic Description of the Mint Plant ‘Kushal’

[0016] 1. Genus: Mentha.

[0017] 2. Species: arvensis L.

[0018] 3. Family: Lamiaceae.

[0019] 4. Common name Japanese mint/corn mint/menthol mint.

[0020] 5. Plant height 70.0 to 108.0.

[0021] 6. Plant canopy 83-85 cm.

[0022] 7. Growth habit: Erect sturdy main stem, profuse branching.

[0023] 8. Stem: Round to quadrangular hard, woody, green (144A), 5-11 mmthick at 5^(th) internode.

[0024] 9. Leaf:

[0025]Colour.—Light green (137B).

[0026]Texture.—Moderately thick and rigid.

[0027]Surface.—Hairy and rough.

[0028]Shape.—Lacerate.

[0029]Margin.—Moderately deep serration (18 to 50 number).

[0030]Tip.—Acute.

[0031]Base.—Attenuate.

[0032]Size.—Moderately broad.

[0033]Petiole length.—1.5±0.1.

[0034]Area.—6.0 cm² (Average of full branch).

[0035]Length.—7.75±0.3.

[0036]Width.—4.8±0.4.

[0037] 10. Leaf:stem ratio (w/w): 0.88 to 1.54.

[0038] 11. Inflorescence: Indefinite recemose.

[0039]Total number of florets.—20 to 40.

[0040] 12. Flowers: Arranged in whorls surrounding the stem at the baseof lateral leaves.

[0041]Flower length (mm).—3.50.

[0042]Pedicel.—Yellow green (145C).

[0043]Calyx.—Four, Yellow green (143C).

[0044]Corolla.—Pinkish white, four, fused to a bell shaped corolla tube(56C).

[0045]Anthers.—Four, ocidimetary, come out of the corolla tube.

[0046]Stigma.—Bifid, Purple (76A).

[0047] 13. Oil content in the fresh herb (%): 0.7 to 0.9.

[0048] 14. Oil quality:

[0049]Menthol content (%).—77 to 81.

[0050]Congealing point.—20 to 21° C.

[0051] 15. Herbage (Shoot biomass Q/100 m²): 1.72 to 3.33.

[0052] In addition to producing more herbage and essential oilcomparatively, the plant ‘Kushal’ adopts quickly to late plantingcondition. Water logging condition during sucker production does nothamper the survival, growth and proliferation of the plant. The plant ofinvention ‘Kushal’ produces herbage comparable to the improved variety‘Kosi’ if planted in the month of January (normal date ofplanting)(Table 1). But in late planting during April the plant ‘Kushal’surpasses the growth, yield of herbage and oil to any other existingvarieties (table 2). Kushal produces more herbage and more essential oilcompared to the most improved variety ‘Kosi’ (now cultivated widely inmint growing regions of India) when planted late.

[0053] From the parent plant ‘Himalaya’ the new plant of invention isfar ahead in terms of herbage and essential oil yield when planted ineither January or April. So, as per the objective we could select aplant type which can be planted in the month of April if the main fieldafter the harvesting of Rabi crop. Generally the land after the harvestof wheat, chickpea is left as fallow till the next planting of rice cropduring rainy season (Khariff). Otherwise a short duration crop isplanted within this period which may yield low income to the farmer.Instead if a crop of mint is harvested during this period the farmers orthe landowner will be benefited immensely as mint is a cash crop.Improved plant type with adaptability to late planting combined withbetter yield of herbage, essential oil and ultimately menthol in theplant of invention ‘Kushal’ can enhance the income of the planter inlong run. TABLE 3 Additional description of ‘Kushal’ compared to theparent ‘Himalaya’. Character cv.Himalaya cv Kushal 1. Leaf:stem ratio 1.0  1.54 2. Stem colour Green(143C) Green (144A) lower purplish(70A)Stiffness Hard Hard Thickness at 5^(th)  8.0 11 internode(mm) 3. LeafColour Green(138B) Green (137B) Length(cm)  6.2  8.0 Width(cm)  3.6  5.2Area(cm²) 15.4 15.6 4. Petiole length (cm)  1.5  2.2 5. Flower colourPinkish white Pinkish white (56C) 6. Flower length (mm)  3.72  3.50 7.Calyx colour Green (143C) Green (143C) 8. Stigma colour White purplishwhite (76A) 9. Disease incidence to Rust Resistant Resistant Alternarialeaf blight Resistant Resistant Corynespora leaf spot Tolerant TolerantPowdery mildew Tolerant Tolerant

[0054] Evidence of Uniformity and Stability

[0055] No variants of any kind (morphological or molecular) have beenobserved since 1998 (the year of development) and through yield trialsin the following years indicating the stability and uniformity of thegenotype. Further, the comparative total herbage, oil and ultimatelymenthol yields of ‘Kushal’ were significantly higher in comparison tothe parent variety ‘Himalaya’, the high menthol yielding variety Kalka(CIMAP/HY77), and best variety check ‘Kosi’ in different years andseasons. The traits of improved herbage, essential oil yield and theadaptability to late transplanting is unprecedented and stable.

[0056] Statement of Distinction

[0057] The genotype ‘Kushal’ possessing the traits of increasedadaptability to late transplanting combined with higher essential oiland herbage yield, is unique and unprecedented not possessed by anyknown variety. The genotype is having higher biomass and higher oilyield unit area in comparison to others. Its genetic make up is distinctin terms of DNA profile.

[0058] The colour codes are in accordance with the “RHS colour chartpublished by The Royal Horticultural Society, 80 Vincent Square, LondonSW1P 2PE,1995.

[0059] Randomly Amplified Polymorphic DNA Analysis:

[0060] The RAPD profiles of the plant ‘Kushal’ were unambiguously ableto establish its distinct identity as completely different from theparent plant ‘Himalaya’ as well as the known released varieties. Theplant of the present invention was developed by screening molecularvariants among somaclones already differentiated as distinct, unique andnovel at DNA level. The plant is having desirable morphological andeconomical traits in a rare unmatchable combination and is availableonly with us at CIMAP. No variation in the RAPD patterns was observed inthe analysis of the micropropagated as well as field raised populationin successive generations indicating the stability of the genotype. The20 MAP primers (MAP 01 to MAP 20) synthesized in the laboratory usingABI 392 DNA synthesizer, with the sequence AAATCGGAGC, GTCCTACTCG,GTCCTTAGCG, TGCGCGATCG, AACGTACGCG, GCACGCCGGA, CACCCTGCGC, CTATCGCCGC,CGGGATCCGC, GCGAATTCCG, CCCTGCAGGC; CCAAGCTTGC, GTGCAATGAG, AGGATACGTG,AAGATAGCGG, GGATCTGAAC, TTGTCTCAGG, CATCCCGAAC, GGACTCCACG, AGCCTGACGCwere used for the analysis to develop the unique fingerprint pattern.Accordingly the invention provides a new and distinct mint plant ofMentha arvensis ‘Kushal’, developed through tissue culture, possessingthe following combination of characters:

[0061] a. the said plant is able to adopt quickly to late plantingcondition and produce higher herbage and essential oil compared to theexisting varieties,

[0062] b. the plant is suitable for transplanting using grown shoots inApril as well as normal planting through suckers in the month February,

[0063] c. the said plant produces high essential oil yield (0.7 to 0.9%)as well as herbage yield (1.72 to 3.33 Q per 100 m²),

[0064] d. the said plant possesses better growth and vegetative growthwith high regenerability covering at least 83-85 cm canopy area and aheight of at least 70.0 to 108.0 cm in a maximum of 110 days,

[0065] e. the said plant has distinct molecular profile by randomamplified polymorphic DNA (RAPD) using 20 random primers distinguishingthe plant from the other existing varieties,

[0066] f. the said plant retains the characteristics of tolerance toleaf spot, rust and powdery mildew as in the parent variety ‘Himalaya’,

[0067] g. the said plant has light greenish leaves(138B), pinkish whiteflowers (56C) like the parent plant ‘Himalaya’ but green stem (144A),

[0068] h. the plant genotype ‘Kushal’ withstand waterlogging conditionduring sucker production in terms of survival, growth compared to othervarieties checked.

1 20 1 10 DNA Artificial Sequence Description of Artificial SequenceSynthetic primer 1 aaatcggagc 10 2 10 DNA Artificial SequenceDescription of Artificial Sequence Synthetic primer 2 gtcctactcg 10 3 10DNA Artificial Sequence Description of Artificial Sequence Syntheticprimer 3 gtccttagcg 10 4 10 DNA Artificial Sequence Description ofArtificial Sequence Synthetic primer 4 tgcgcgatcg 10 5 10 DNA ArtificialSequence Description of Artificial Sequence Synthetic primer 5aacgtacgcg 10 6 10 DNA Artificial Sequence Description of ArtificialSequence Synthetic primer 6 gcacgccgga 10 7 10 DNA Artificial SequenceDescription of Artificial Sequence Synthetic primer 7 caccctgcgc 10 8 10DNA Artificial Sequence Description of Artificial Sequence Syntheticprimer 8 ctatcgccgc 10 9 10 DNA Artificial Sequence Description ofArtificial Sequence Synthetic primer 9 cgggatccgc 10 10 10 DNAArtificial Sequence Description of Artificial Sequence Synthetic primer10 gcgaattccg 10 11 10 DNA Artificial Sequence Description of ArtificialSequence Synthetic primer 11 ccctgcaggc 10 12 10 DNA Artificial SequenceDescription of Artificial Sequence Synthetic primer 12 ccaagcttgc 10 1310 DNA Artificial Sequence Description of Artificial Sequence Syntheticprimer 13 gtgcaatgag 10 14 10 DNA Artificial Sequence Description ofArtificial Sequence Synthetic primer 14 aggatacgtg 10 15 10 DNAArtificial Sequence Description of Artificial Sequence Synthetic primer15 aagatagcgg 10 16 10 DNA Artificial Sequence Description of ArtificialSequence Synthetic primer 16 ggatctgaac 10 17 10 DNA Artificial SequenceDescription of Artificial Sequence Synthetic primer 17 ttgtctcagg 10 1810 DNA Artificial Sequence Description of Artificial Sequence Syntheticprimer 18 catcccgaac 10 19 10 DNA Artificial Sequence Description ofArtificial Sequence Synthetic primer 19 ggactccacg 10 20 10 DNAArtificial Sequence Description of Artificial Sequence Synthetic primer20 agcctgacgc 10

1. A new and distinct mint plant of Mentha arvensis ‘Kushal’, developedthrough tissue culture, possessing the following combination ofcharacters: a. the said plant is able to adopt quickly to late plantingcondition and produce higher herbage and essential oil compared to theexisting varieties, b. the plant is suitable for transplanting usinggrown shoots in April as well as normal planting through suckers in themonth February, c. the said plant produces high essential oil yield (0.7to 0.9%) as well as herbage yield (1.72 to 3.33 Q per 100 m²), d. thesaid plant possesses better growth and vegetative growth with highregenerability covering at least 83-85 cm canopy area and a height of atleast 70.0 to 108.0 cm in a maximum of 110 days, e. the said plant hasdistinct molecular profile by random amplified polymorphic DNA (RAPD)using 20 random primers distinguishing the plant from the other existingvarieties, f. the said plant retains the characteristics of tolerance toleaf spot, rust and powdery mildew as in the parent variety ‘Himalaya’,g. the said plant has light greenish leaves(138B), pinkish white flowers(56C) like the parent plant ‘Himalaya’ but green stem (144A), h. theplant genotype ‘Kushal’ withstand waterlogging condition during suckerproduction in terms of survival, growth compared to other varietieschecked.